PROJECT ABSTRACT/SUMMARY The hepatitis C virus (HCV) is a human hepatotropic hepacivirus known for causing persistent infections that often progress to fatal liver diseases. Although new antivirals cure >97% of infections, the vast majority of infected patients are either undiagnosed or unable to receive therapy due to substantial socioeconomic barriers. To achieve global eradication, a vaccine to prevent HCV persistence will be needed. Although the immune factors that dictate whether an infection will be controlled are poorly defined, T cells are considered key immunological determinants. Indeed, silencing of virus-specific CD8+ T cells is a classic feature of infections that persist. Furthermore, the only vaccine to advance to phase II clinical testing in humans is based on a T cell immunization strategy that showed signs of efficacy in the chimpanzee model. Despite this promising momentum, it is not fully clear why this approach succeeded in chimps and whether similar results can be expected in humans. Indeed, mechanisms of vaccine-induced immunity are incompletely known and deciphering these in humans will be challenging and slow. Furthermore, due to the elimination of chimps from research, there are no suitable animal models available to further evaluate vaccine-mediated protection against HCV persistence. Recently, we developed a surrogate model for HCV using a homologous hepacivirus found in feral rats. Several aspects of this model are significant. First, infection in lab rats is strictly hepatotropic and highly persistent despite the activation of host adaptive immunity. Second, immune responses can be readily analyzed in the infected liver, which is poorly accessible in humans. Finally, use of a rodent host permits the expedited testing and mechanistic deconstruction of promising vaccination concepts, which was not easily afforded by chimps. Our long-term goal in this proposal is to identify correlates of vaccine-induced immunity to chronic hepacivirus infection and to translate this knowledge into rational HCV vaccine designs. As the impetus for our studies, we show in our preliminary data that persistent infection in rats can be prevented using an adenovirus-vectored vaccine encoding non-structural proteins, which primes cellular immunity in the absence of neutralizing antibodies. How this approach leads to control of an otherwise highly persistent infection is unclear. Our central hypothesis is that CD8+ T cell function is essential for hepacivirus clearance, and pre-exposure priming of CD4+ T cell help via vaccine protects virus-specific CD8+ T cells from failure during infection, resulting in effective antiviral control. Two specific aims are proposed. Aim 1 will provide a detailed comparison of cellular immunity between vaccinated and unvaccinated animals after virus challenge; functional responses will be comprehensively analyzed and minimal T cell epitopes will be mapped. Aim 2 will test the requirement for CD4+ and CD8+ T cells in vaccine-induced protection; cell depletion and active/passive vaccination experiments will be performed. Our overall results from this proposal will have major implications for HCV vaccine and immunotherapy development in humans, where experiments of this type are not possible.